In the recent past, light-microscopic imaging methods have been developed with which, based on a sequential, stochastic localization of individual markers, in particular fluorescence molecules, samples can be imaged that are smaller than the diffraction resolution limit of conventional light microscopes. Such methods are described, for example, in WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1, U.S. 2009/0134342 A1; DE 10 2008 024 568 A1; “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)”, Nature Methods 3, 793-796 (2006), M. J. Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching”, Geisler C. et al, Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as localization microscopy. The applied methods are known in the literature, for example, under the designations (F)PALM ((Fluorescence) Photoactivation Localization Microscopy), PALMIRA (PALM with Independently Running Acquisition), GSD(IM) (Ground State Depletion Individual Molecule return) Microscopy) or (F)STORM ((Fluorescence) Stochastic Optical Reconstruction Microscopy).
The new methods have in common that the samples to be imaged are prepared with markers that have two distinguishable states, namely a “bright” state and a “dark” state. When, for example, fluorescent dyes are used as markers, then the bright state is a state in which they are able to fluoresce and the dark state is a state in which they are not able to fluoresce. In order to image a sample with a resolution that is higher than the conventional resolution limit of the imaging optics, a small subset of the markers is repeatedly switched to the bright state. This “active” subset forms a marker pattern whose individual markers, which have been switched to the bright state, have an average distance from each other greater than the resolution limit of the imaging optics. The respective marker pattern is then imaged onto a spatially resolving image sensor which captures the individual markers in the form of spatially separable light distributions.
In this way, a plurality of individual images are captured, in each of which a different marker pattern is depicted. In an image analysis process, then in each individual image, the positions of the centroids of the light distributions are determined, which represent the markers that are in their bright state. The centroid positions of the light distributions determined from the individual raw data images are then combined into one representation in the form a complete image. The high-resolution complete image produced by this combined representation reflects the distribution of the markers.
In order to obtain a representative image of the sample to be imaged, a sufficient number of marker signals must be detected. However, since the number of markers in the particular active marker pattern is limited by the average minimum distance by which two markers in the bright state must spaced from each other, it is necessary to capture a very large number of individual images in order to produce a complete image of the sample. Typically, the number of individual images is in a range from 10,000 to 100,000.
The time required to capture an individual image cannot be less than a lower limit determined by the maximum image acquisition rate of the image sensor. This results in relatively long total imaging times for a sequence of individual images needed for a complete image. The total imaging time may, for example, be up to several hours.
During a total imaging time of such a length, thermal effects, such as thermal expansion, contraction or strain of the mechanical components of the light microscope may result in a drift of the sample to be imaged relative to the imaging optics. Since in order to create a complete high-resolution image, all individual images are combined after the determination of the centroids, any relative movement between the sample and the imaging optics that may occur between the acquisition of two successive individual images will degrade the spatial resolution of the complete image.